Hydroxyl group containing polymers such as polysaccharide gels are widely used as solid supports for preparing biospecific affinity matrices. Several methods are known for coupling biologically active affinity ligands to water insoluble carriers containing hydroxyl groups. These methods have been used for binding proteins such as enzmes, antibodies and antigens to solid carriers to produce coupling products which have found use in many different fields of technology. One example thereof is in connection with immunologic determination methods wherein antibodies or antigens have been bonded to water insoluble polymeric carriers. Another important application is in connection with affinity chromatography wherein an organic ligand having biospecific affinity to some other organic substance has been bonded to water insoluble polymeric carriers. Water insoluble polymers have also been bonded to proteins for modifying properties thereof.
The coupling of the ligand to the carrier is often carried out in such a manner that the carrier is activated by a reactive group which is then reacted with the desired ligand. Examples of known activation methods are activation by means of cyanogen bromide, CNBr, which is disclosed in Porath, et al., "Immobilized Enzymes. Methods in Enzymology," Vol. 44 (Mosbach, K., Ed.) page 19-45, Academic Press, New York (1976). The use of CNBr for activating hydroxyl groups of polymeric carriers is the earliest and most widely used method. However, CNBr activation procedures suffer from certain disadvantages, namely, (1) the linkages formed between CNBr activated gels and amino groups of affinity ligands are labile, (2) the reaction results in the introduction of charged species which interfere with affinity absorption, and (3) CNBr is a noxious, lachrimatory and poisonous chemical which requires special care in its handling. Another method for activating polymeric carriers is the use of organic sulfonyl chlorides, particularly, 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). Such activating agents are, however, relatively expensive and are in liquid rather than solid forms. Further, some prior art coupling methods may give rise to changed charge conditions for the organic ligand, for example, by the formation of charged groups at their binding site during the coupling. This makes the product unsuitable for affinity chromatography.
The present invention has as a principal object providing a stable and hydrolysis-resistant coupling product of a polymeric gel and an organic ligand in which the organic ligand is covalently bonded directly to a carbon atom in the polymeric carrier gel. Another object of the present invention is to provide a process which can be conducted under relatively mild conditions in order to avoid any detrimental effect upon reactants such as sensitive biological ligands.
These and other objects of the invention are achieved by forming a reactive derivative of a polymeric hydroxyl-containing gel by reacting the gel with 2-fluoro-1-methylpyridinium toluene-4-sulfonate, which will hereinafter be referred to at times as FMP, and then reacting the activated carrier with a ligand containing a primary amino or sulfhydryl group.
The method according to the present invention thus comprises an activation step, wherein the 1-methyl-2-pyridoxy type leaving group is introduced into the polymeric carrier and a coupling step in which the organic ligand is bonded covalently to the polymeric character while splitting off the leaving group.